Process for the preparation of glycosylated interferon beta

Abstract

The present invention relates to a process for the production of interferon beta, and to an interferon beta composition having a unique glycosylation pattern.

We claim: 
     
       1. A process for the manufacturing of glycosylated recombinant human interferon beta, comprising culturing a human interferon beta producing cell in a serum-free medium, said culturing comprising a growth phase I, a growth phase II and a production phase, wherein growth phase I is carried out at about 37° C., growth phase II is carried out at about 35° C., and the production phase is carried out at about 33° C., said serum-free medium comprising:
 a) about 10 to about 30 mM HEPES; 
 b) about 0.5 to about 3 mM Proline; 
 c) about 5500 to about 7000 mg/L sodium chloride; and 
 d) N-acetyl cysteine alone or in combination with zinc. 
 
     
     
       2. The process according to  claim 1 , said serum-free medium comprising:
 a) about 10 to about 30 mM HEPES; 
 b) about 0.5 to about 3 mM Proline; and 
 c) about 5500 to about 7000 mg/L sodium chloride: and 
 d) N-acetyl c swine alone. 
 
     
     
       3. The process according to  claim 2 , said serum-free medium further comprising about 10 to about 20 mg/L phenol red. 
     
     
       4. The process according to  claim 1 , wherein the process is a perfusion process with a dilution rate ranging from about 1 to about 10. 
     
     
       5. The process according to  claim 4 , wherein the dilution rate is increased within the first two to three weeks of cell culture from an initial value of about 1 to 2 per day to a value of about 7 to 10 per day. 
     
     
       6. The process according to  claim 1 , further comprising:
 a) subjecting the medium containing human interferon beta to affinity chromatography and eluting said human interferon beta; 
 b) subjecting the human interferon beta containing eluate to cation exchange chromatography and eluting said human interferon beta; and 
 c) subjecting the eluate of the cation exchange chromatography to hydrophobic chromatography by RP-HPLC and eluting said human interferon beta. 
 
     
     
       7. The process according to  claim 6 , comprising, before step (a), clarifying of the medium by filtration. 
     
     
       8. The process according to  claim 7 , further comprising:
 d) performing ultrafiltration and dialysis; 
 e) subjecting the dialysate to size exclusion chromatography; and 
 f) subjecting the eluate of the size exclusion chromatography to microfiltration. 
 
     
     
       9. The process according to  claim 6 , wherein step (a) is carried out on Blue Sepharose and step (b) is carried out on Carboxymethyl Sepharose. 
     
     
       10. The process according to  claim 1 , wherein said human interferon beta producing cell comprises:
 a) a nucleic acid comprising a human interferon beta coding sequence functionally linked to a SV40 T Ag early polyadenylation region, wherein the nucleic acid does not comprise the human interferon beta polyadenylation signal; 
 b) a nucleic acid comprising a human interferon beta coding sequence functionally linked to a SV40 T Ag early polyadenylation region, wherein the nucleic acid does not comprise the human interferon beta polyadenylation signal and wherein said nucleic acid does not comprise the human interferon beta 3′ UTR; 
 c) a nucleic acid comprising a SV40 promoter/enhancer functionally linked to a human interferon beta coding sequence, wherein the human interferon beta coding sequence is functionally linked to the SV40 T Ag early polyadenylation region and the nucleic acid does not comprise the human interferon beta polyadenylation signal or the human interferon beta 3′ UTR; 
 d) a nucleic acid according to a), b) or c), wherein said nucleic acid further comprises a mouse dihydrofolate reductase (DHFR) gene; 
 e) a nucleic acid according to d), wherein said mouse DHFR gene is functionally linked to a SV40 T Ag polyA-containing early polyadenylation region; or 
 f) a nucleic acid according to e), further comprising a SV40 promoter/enhancer functionally linked to the mouse DHFR gene. 
 
     
     
       11. The method according to  claim 1 , wherein growth phase I is carried out at about 37° C. until the glucose consumption rate (GCR) is greater than or equal to 2.0±1.0 grams per liter per day (g·L −1 ·d −1 ). 
     
     
       12. The method according to  claim 1 , wherein growth phase II is carried out at about 35° C. until the GCR is greater than or equal to 8.0±0.5 grams per liter per day (g·L −1 ·d −1 ). 
     
     
       13. The method according to  claim 1 , wherein growth phase I is carried out at about 37° C. until the glucose consumption rate (GCR) is greater than or equal to 2.0±1.0 grams per liter per day (g·L −1 ·d −1 ) and growth phase II is carried out at about 35° C. until the GCR is greater than or equal to 8.0±0.5 grams per liter per day (g·L −1 ·d −1 ). 
     
     
       14. The method according to  claim 1 , wherein said serum-free medium comprises 20 mM HEPES, 1 mM proline, 15 mg/L phenol red and 6150 mg/L NaCl. 
     
     
       15. The method according to  claim 1 , said serum-free medium comprising:
 a) about 10 to about 30 mM HEPES; 
 b) about 0.5 to about 3 mM Proline; 
 c) about 5500 to about 7000 mg/L sodium chloride; and 
 d) N-acetyl cysteine in combination with zinc. 
 
     
     
       16. The method according to  claim 1 , wherein said serum-free medium comprises 20 mM HEPES, 1 mM proline, 15 mg/L phenol red, 6150 mg/L NaCl and N-acetyl cysteine. 
     
     
       17. The method according to  claim 1 , wherein said serum-free medium comprises 20 mM HEPES, 1 mM proline, 15 mg/L phenol red, 6150 mg/L NaCl and N-acetyl cysteine in combination with zinc.